Novel biomarker for predicting sensitiviy to egfr-targeting agent, and use thereof

ABSTRACT

The present disclosure relates to a new biomarker for predicting susceptibility to an EGFR-targeted agent and a use thereof, and more particularly, provides a biomarker for predicting susceptibility to an EGFR (Epidermal Growth Factor Receptor)-targeted agent, comprising a RON (Recepteur d&#39; Origine Nantais) gene; a composition for predicting susceptibility to the EGFR-targeted agent, comprising an agent which measures a gene expression level of the biomarker; or an expression or activity level of a protein thereof; a composition for enhancing the susceptibility to the EGFR-targeted agent, comprising an inhibitor of the expression of the gene or the expression or activity of the protein of the gene as active ingredients; a kit for predicting the susceptibility to the EGFR-targeted agent, comprising the composition; and a method for predicting the susceptibility to the EGFR-targeted agent. According to the present disclosure, since an effect of predicting the susceptibility to the EGFR-targeted agent is excellent in a colon cancer, the present disclosure may be useful in the treatment of the colon cancer.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/329,942, filed on Jan. 27, 2017, which is a United States NationalPhase entry of International Application No. PCT/KR2015/007964 filedJul. 29, 2015, which claims the benefit of Korean Patent Application No.10-2014-0096716 filed Jul. 29, 2014. The entire contents of each of theforegoing applications is hereby incorporated by reference herein.

TECHNICAL FIELD

The present disclosure is achieved by Project No. 1420030 under thesupport of the Ministry of Health of Korea and a research managementspecialized organization of the project is Research Supporting Programfor Regional Cancer Center, a research program name is “National R&DProgram for Cancer Control”, a research project name is “Development ofNew Therapy for Overcoming Centuximab Resistance in Colon Cancer Patientand Development of Biomarker”, a control organization is Asan MedicalCenter, and a research period is May 1, 2014 to Apr. 30, 2017.

The present disclosure is achieved by Project No. HI06C0868 under thesupport of the Ministry of Health of Korea and the research managementspecialized organization of the report is Korea Health IndustryDevelopment Institute, the research program name is “LeadingCharacterization Research Program”, the research project name is“Development of Creative Anticancer Drug Targeting Receptor TyrosineKinase (RTK)”, the control organization is Leading Cancer ResearchBusiness Group of Asan Medical Center, and the research period is Dec.1, 2011 to Nov. 30, 2016.

The present disclosure relates to a new biomarker for predictingsusceptibility to an EGFR-targeted agent and a use thereof.

BACKGROUND

Generally, in an anticancer therapy, a biological response whenadministering an anticancer agent significantly depends onsusceptibility to the anticancer agent of cancer cells targeted to theanticancer agent. The susceptibility of the cancer cell to theanticancer agent significantly varies for each cancer cell. Thedifference in susceptibility is caused due to a quantitative orqualitative difference of a target molecule of the cancer agent or afactor associated therewith or acquisition of drug-resistance. Based onthe background, when a targeted cancer cell exhibits the susceptibilityto the drug, if a genetic change of the cancer cell specificallyexhibited may be verified, determination of an effect of the drug,establishment of the therapy, selection of a new therapy and the likeare valid, and as a result, it is greatly beneficial. Further, in acancer tissue obtained by a bio tissue piece, and the like prior totherapy, the cancer cell is separated and drug processing is performedaccording to a general method, and as a result, when whether the cancelcell is sensitive to the drug is measured by the change, whether thetherapy by the drug is valid may be predicted, it is very usefulclinically.

In general, it was known that mutation of a KRAS, NRAS, or BRAF genemakes protein having a signaling characteristic deformed from the cancercell and the mutation is associated with an unsuccessful result in acancer therapy using a therapeutic antibody targeting an epidermalgrowth factor receptor, for example, cetuximab or panitumumab (Amado,Wolf et al, 2008; Karapetis, Khambata-Ford et al, 2008; Di Nicolantonio,Martini et al, 2008; Loupakis, Ruzzo et al, 2009; Lievre, Bachet et al,2006).

However, even in the case of a cancer patient having not mutation but aKRAS, NRAS, or BRAF wild type genotype, there are many cases in which ananticancer effect of a target anticancer agent such as cetuximab, or thelike is less.

Cetuximab as a recombination anti-EGFR human/mouse chimeric monoclonalantibody (MoAb) was known to sensitize the cancer cell toantibody-dependent cell toxicity, and chemotherapy and radiotherapy(Graham J, et al., Nat Rev Drug Discov. July 2004; 3(7):549-50; KimuraH. et al., Cancer Sci. August 2007; 98(8):1275-80; Kurai J, et al., ClinCancer Res. Mar. 1, 2007; 13(5):1552-61; Dittmann K. et al.,RadiotherOncol, August 2005; 76(2):157-61.). Due to the advantage, aclinical advantage using cetuximab as a single therapy or a concurrenttherapy of chemotherapy and/or radiotherapy has been proved in headcancer and cervical cancer and metastatic colon cancer (Marshall J, etal., Cancer. Sep. 15, 2006; 107(6):1207-18).

Meanwhile, Recepteur d' Origine Nantais (RON) as a protein receptor thatbelongs to c-MET series is a receptor of macrophage-stimulating protein(MSP) secreted from liver and controlling an operation of macrophagocyte(Zhou Y Q, He C, Chen Y Q, Wang D, Wang M H: Altered expression of theRON receptor tyrosine kinase in primary human colorectaladenocarcinomas, generation of different splicing RON variants and theironcogenic potential, Oncogene 2003, 22(2):186-197). Expression of RON isabnormally controlled in breast cancer and colorectal cancer and inparticular, is closed related with metastasis of colorectal cancer. Forexample, as it is reported that a single clone antibody IMC-41A10 bondedto RON inhibits cell metastasis and cancer formation, a RON inhibitorwill be able to exhibit an excellent effect in anti-cancer and cancermetastasis.

That is, an anti-cancer drug exhibits a large difference amongindividuals in terms of resistance and toxicity and since theanti-cancer drug exhibits resistance over the half number even in thesame patient, and as a result, selection using an appropriatetherapeutic responsiveness marker may bring about a remarkable advancein anti-cancer drug therapy. Therefore, a research into therapyresponsiveness of individual anti-cancer drugs depending on a specificgene has been continuously actively progressed in recent years.

However, there is no notable accomplishments due to a complex operationof a bio response related element to a specific drug, diversity ofmedicines and administration schemes, and a difficulty in securingenormous samples.

SUMMARY

The present inventors have made intensive efforts to develop the methodfor predicting the susceptibility to the EGFR-targeted agent as theanticancer agent in the colon cancer by analyzing an activity level ofthe RON gene and/or the protein as the biomarker for predicting thesusceptibility in the colon cancer to cetuximab which is one ofEGFR-targeted agents. As results, the present inventors has verifiedthat the cell death degree is different by the drug susceptibility tocetuximab as the EGFR-targeted agent by having an effect on the Adam11,Adam32, FZD4, GPER, and GPR101 genes which areEGFR-transactivation-related genes according to the activity level ofthe RON gene and/or the protein in the colon cancer, thereby completingthe present disclosure.

Accordingly, it is an object of this invention to provide a biomarkerfor predicting susceptibility to an EGFR-targeted agent.

It is another object of this invention to provide a composition forpredicting susceptibility to an EGFR-targeted agent.

It is yet another object of this invention to provide a composition forpredicting susceptibility to an EGFR-targeted agent.

It is still another object of this invention to provide a kit forpredicting susceptibility to an EGFR-targeted agent.

It is still yet another object of this invention to provide asusceptibility enhancer to an EGFR-targeted agent.

It is still yet another object of this invention to provide a method forpredicting susceptibility to an EGFR-targeted agent.

It is still yet another object of this invention to provide apharmaceutical composition for preventing or treating cancer, comprisingthe susceptibility enhancer to the EGFR-targeted agent and theEGFR-targeted agent as active ingredients.

It is still yet another object of this invention to provide a method forenhancing susceptibility to the EGFR-targeted agent, comprisingadministering the susceptibility enhancer to the EGFR-targeted agent andthe EGFR-targeted agent to a subject.

According to the embodiment of the present disclosure, when thebiomarker for predicting the susceptibility to the EGFR-targeted agentis used, the susceptibility of an individual patient may be accuratelydetermined before treatment initiation, and thus, it is possible toselect an anticancer agent having a high treatment effect. Further,since the use of the anticancer agent without obtaining the effect isavoided, it is possible to avoid unnecessary side effects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates presence or absence of activation of RON proteins ina human colon cancer cell line.

FIG. 1B illustrates an analysis result of genes changed by only RONgenes.

FIG. 1C illustrates a real-time PCR analysis result of genes relatedwith EGFR transactivation by RON activation.

FIG. 1D illustrates an RT-PCR expression analysis result of genesrelated with EGFR transactivation by RON activation.

FIG. 1E illustrates a real-time PCR analysis result of genes relatedwith EGFR transactivation by RON inhibition.

FIG. 1F illustrates an analysis result of activation of EGFR proteins byRON activation.

FIG. 2A illustrates an induced phosphorylation result of EGFR by RONactivation.

FIG. 2B illustrates an analysis result of a change in activation of EGFRproteins by RON inhibition.

FIG. 2C illustrates an analysis result of a change in EGFRligand-independent EGFR activation by RON inhibition.

FIG. 3A illustrates an analysis result of endogenous intracellularbinding between the RON protein and the EGFR protein.

FIG. 3B illustrates an analysis result of exogenous intracellularbinding between the RON protein and the EGFR protein.

FIG. 3C illustrates an analysis result of an EGFR protein domain boundwith the RON protein through an in vivo full down assay.

FIG. 3D illustrates an analysis result of an EGFR protein domain boundwith the RON protein through an in vitro cell free full down assay.

FIG. 3E illustrates an analysis result of predicting an RON proteinbinding site bound with the EGFR protein using computer modeling.

FIG. 3F illustrates an analysis result of an RON protein binding sitebound with the EGFR protein.

FIG. 3G illustrates an analysis result of EGFR activation according to abinding site of the RON protein bound with the EGFR protein.

FIG. 4A illustrates an analysis result of presence or absence ofligand-independent EGFR activation according to presence or absence ofan RON active form protein.

FIG. 4B illustrates an analysis result of presence or absence ofligand-independent EGFR activation according to presence or absence ofligand-independent activation of RON.

FIG. 4C illustrates an analysis result of the activity of EGFR uponoverexpression of RON del155, RON del155/K1114M, and EGFR.

FIG. 5A illustrates an analysis result of inhibiting EGFR activationthrough inhibition of RON activation and inhibiting EGFR activation tocetuximab.

FIG. 5B illustrates an analysis result of cell death by cetuximabaccording to presence or absence of RON activation.

FIG. 5C illustrates an analysis result of cell death by cetuximab afterinhibiting RON expression.

FIG. 5D illustrates an analysis result of a sub-signaling mechanism bycetuximab after inhibiting RON expression.

FIG. 5E illustrates an analysis result of cell death by cetuximab afterinhibiting RON activation.

FIG. 5F illustrates an analysis result of cell death by cetuximabaccording to presence or absence of RON activation.

FIG. 5G illustrates an analysis result of inhibiting a cell growth bycetuximab according to presence or absence of RON activation.

FIG. 5H illustrates an analysis result of cell death by cetuximab in aRON knockout isogenic cell line.

FIG. 5I illustrates an analysis result of cell growth inhibition bycetuximab in a RON knockout isogenic cell line.

FIG. 6 illustrates an analysis result of a tumor inhibition effect ofcetuximab according to presence or absence of RON in an in vivoxenograft model using a RON knockout isogenic cell line.

FIG. 7 illustrates a result of treating both an ROM antibody anticanceragent and cetuximab in a cell line having RON activation.

FIG. 8 illustrates a result of analyzing RON activation in tissue fromcolon cancer patient(treated with cetuximab).

FIG. 9 is a table illustrating that it was known that the KM12C coloncancer cell line had the KRAS, NRAS, and BRAF wild type genes and hadresistance to cetuximab (the left-most column indicating BRAF V600Emutation and the adjacent column to the right indicating KRAS mutation).

DETAILED DESCRIPTION

Hereinafter, the present disclosure will be described in more detail.

In one aspect of the present invention, there is provided a biomarker ora biomarker composition for predicting susceptibility to an epidermalgrowth factor receptor (EGFR)-targeted agent including a recepteur d'origine nantais (RON, NM_002447.1) gene.

The largest feature of the present disclosure is to predictsusceptibility to the EGFR-targeted agent using activation of the RONgene and an activated protein which is a product thereof as a biomarker.

The biomarker of the present disclosure may be an indicator ofsusceptibility to an anticancer agent which is the EGFR-targeted agentand may be used in treatment of generation, development, and/ormetastasis of the cancer due to excellent accuracy and reliability as asensitive marker to the anticancer agent.

The term “susceptibility” used in this specification means whether tohave an effect on a specific drug for the cancer of a subject patient.

The term “susceptibility” used while mentioning the susceptibility inthis specification means that an effect on the drug is acted bysusceptibly reacting with the corresponding drug and is mixed with thesusceptibility in this specification.

The term “resistance” used while mentioning the susceptibility in thisspecification means that the effect on the drug is not acted withoutsusceptibly reacting with the corresponding drug.

For example, the specific drugs are mainly anticancer agents, and theanticancer agents have the effect on the cancer or not depending on thetype of cancer. Further, in spite of a kind of cancer that is recognizedas valid, it is known that there are a case with the effect and a casewithout the effect according to a target subject patient. Whether theanticancer agent has an effect on the cancer of the target patient isreferred to as susceptibility to the anticancer agent. Accordingly,according to the present disclosure, if patients (responders) that mayexpect the effect before treatment initiation and patients(non-responders) which may not expect the effect can be predicted,chemotherapy with high effectiveness and safety may be implemented.

The term “prediction” used in this specification is used to indicatepossibility of advantageously or disadvantageously responding to a drugor a drug set of the target patient. In an embodiment, the predictionrelates to the degree of the response. For example, the predictionrelates to presence or absence of survival without cancer recurrenceand/or probability thereof after treatment of the patient, for example,treatment of a specific therapeutic agent and/or surgical removal of aprimary tumor and/or chemotherapy for a predetermined period. Theprediction of the present disclosure may be clinically used to determinethe treatment by selecting the most appropriate treatment method for thecolon cancer patient. The prediction of the present disclosure is auseful tool for predicting whether the patient advantageously respondsto a therapeutic treatment, for example, a given therapeutic treatment,such as administration, surgical intervention, chemotherapy of a giventherapeutic agent or a combination thereof or whether long-term survivalof the patient after the therapeutic treatment is possible.

According to a preferred embodiment of the present disclosure, thebiomarker of the present disclosure further includes one or more genesselected from the group consisting of KRAS (V-Ki-ras2 Kirsten ratsarcoma viral oncogene homolog, Gene Bank accession No. NM_033360.2),NRAS (neuroblastoma RAS viral (v-ras) oncogene homolog, Gene Bankaccession No. NP_002515.1), BRAF (v-raf murine sarcoma viral oncogenehomolog B, Gene Bank accession No. NP_004324.2), EGFR (Epidermal GrowthFactor Receptor, Gene Bank accession No. U48722.1), Adam11 (ADAMmetallopeptidase domain 11, Gene Bank accession No. NM_002390.4), Adam32(ADAM metallopeptidase domain 32, Gene Bank accession No. NM_145004.5),FZD4 (Frizzled family receptor 4, Gene Bank accession No. NM_012193.2),GPER (G protein-coupled estrogen receptor 1, NM_001505.2), and GPR101 (Gprotein-coupled receptor 101, Gene Bank accession No. NM_054021.1)genes.

Further, according to a preferred embodiment of the present disclosure,when the KRAS (V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog,NM_033360.2), NRAS (neuroblastoma RAS viral (v-ras) oncogene homolog,NP_002515.1), or BRAF (v-raf murine sarcoma viral oncogene homolog B,NP_004324.2) gene is a wild type, the biomarker of the presentdisclosure predicts the susceptibility to the EGFR-targeted agent andmay be applied to a case where cancer cells with KRAS, NRAS, or BRAFmutation do not achieve a desired effect.

That is, in the present disclosure, the biomarker verifies whether thegenes in the cells are present as the wild type by using the RON, KRAS,NRAS, or BRAF gene and targeting an target having cancer cells or cancerwith the KRAS, NRAS, or BRAF wild-type gene. When the expression levelof the RON gene or the protein expression thereof or the activity levelis low as compared with a normal level (alternatively, the expressionlevel of the wild-type gene/or the protein), the biomarker determinesthat there is the susceptibility to the EGFR-targeted agent. When theexpression level of the RON gene or the protein expression thereof orthe activity level is high as compared with a normal level, thebiomarker determines that there is the resistance to the EGFR-targetedagent.

Further, the Adam11 (ADAM metallopeptidase domain 11, NM_002390.4),Adam32 (ADAM metallopeptidase domain 32, NM_145004.5), FZD4 (Frizzledfamily receptor 4, NM_012193.2), GPER (G protein-coupled estrogenreceptor 1, NM_001505.2), GPR101 (G protein-coupled receptor 101,NM_054021.1) genes are EGFR transcriptional activity-related genes andmay be included as the biomarker that predicts the susceptibility to theEGFR-targeted agent by the RON gene because the expression thereof isinduced according to the activity of the RON gene as the biomarker ofthe present disclosure and the active protein as the product thereof.

Further, the EGFR (Epidermal Growth Factor Receptor, Gene Bank accessionnumber U48722.1) gene may be included as the biomarker that predicts thesusceptibility to the EGFR-targeted agent by the RON gene because theactivation is induced or increased according to the activity of the RONgene as the biomarker of the present disclosure and the active proteinas the product thereof.

In another aspect of the present invention, there is provided acomposition for predicting the susceptibility to the EGFR-targeted agentincluding an agent that measures an expression level of RON (Recepteurd' Origine Nantais, Gene Bank accession number NM_002447.1) gene or anexpression or activity level of the protein thereof.

According to another embodiment of the present disclosure, thecomposition of the present disclosure further includes an agent thatmeasures an expression level of one or more genes selected from thegroup consisting of KRAS (V-Ki-ras2 Kirsten rat sarcoma viral oncogenehomolog, Gene Bank accession No. NM_033360.2), NRAS (neuroblastoma RASviral (v-ras) oncogene homolog, Gene Bank accession No. NP_002515.1),BRAF (v-raf murine sarcoma viral oncogene homolog B, Gene Bank accessionNo. NP_004324.2), EGFR (Epidermal Growth Factor Receptor, Gene Bankaccession No. U48722.1), Adam11 (ADAM metallopeptidase domain 11, GeneBank accession No. NM_002390.4), Adam32 (ADAM metallopeptidase domain32, Gene Bank accession No. NM_145004.5), FZD4 (Frizzled family receptor4, Gene Bank accession No. NM_012193.2), GPER (G protein-coupledestrogen receptor 1, NM_001505.2), and GPR101 (G protein-coupledreceptor 101, Gene Bank accession No. NM_054021.1) genes; or anexpression or activity level of the protein of the genes.

Further, according to a preferred embodiment of the present disclosure,when the KRAS (V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog,NM_033360.2), NRAS (neuroblastoma RAS viral (v-ras) oncogene homolog,NP_002515.1), or BRAF (v-raf murine sarcoma viral oncogene homolog B,NP_004324.2) gene is a wild type, the composition of the presentdisclosure predicts the susceptibility to the EGFR-targeted agent andmay be applied to a case where cancer cells with KRAS, NRAS, or BRAFmutation do not achieve a desired effect.

Further, according to a preferred embodiment of the present disclosure,the EGFR-targeted agent is a therapeutic agent for one or more selectedfrom the group consisting of adrenocorticotropic hormone (ACTH) producedtumors, acute lymphocytic or lymphoblastic leukemia, acute or chroniclymphocytic leukemia, acute non-lymphocytic leukemia, bladder cancer,brain tumors, breast cancer, cervix cancer, chronic myelogenousleukemia, lymphomas, endometriosis, esophagus cancer, bladder cancer,Ewing's sarcoma, tongue cancer, Hopkins lymphoma, Capo sheath sarcoma,kidney cancer, liver cancer, lung cancer, mesothelioma, multiplemyeloma, neuroblastoma, non-hopkin lymphoma, osteosarcoma, ovariancancer, lobular carcinoma, prostate cancer, pancreatic cancer, coloncancer, penis cancer, retinoblastoma, skin cancer, stomach cancer,thyroid cancer, uterine cancer, testicular cancer, wilms tumor, andtrophoblastoma. Further, according to a more preferred embodiment of thepresent disclosure, the EGFR-targeted agent is a therapeutic agent forone or more selected from the group consisting of adrenocorticotropichormone (ACTH) produced tumors, acute lymphocytic or lymphoblasticleukemia, acute or chronic lymphocytic leukemia, acute non-lymphocyticleukemia, bladder cancer, brain tumors, breast cancer, cervix cancer,chronic myelogenous leukemia, lymphomas, endometriosis, esophaguscancer, bladder cancer, Ewing's sarcoma, tongue cancer, Hopkinslymphoma, Capo sheath sarcoma, kidney cancer, liver cancer, lung cancer,mesothelioma, multiple myeloma, neuroblastoma, non-hopkin lymphoma,osteosarcoma, ovarian cancer, lobular carcinoma, prostate cancer,pancreatic cancer, colon cancer, penis cancer, retinoblastoma, skincancer, stomach cancer, thyroid cancer, uterine cancer, testicularcancer, Wilms tumor, and trophoblastoma. According to a still morepreferred embodiment of the present disclosure, the EGFR-targeted agentis a therapeutic agent for colon cancer.

The term “colon cancer” used in this specification means a common nameof rectal cancer, colorectal cancer, and anal cancer.

Further, the EGFR-targeted agent of the present disclosure means ananticancer agent and any EGFR-targeted agent that has an anticancereffect may be applied. Preferably, the EGFR-targeted agent is one ormore selected from the group consisting of cetuximab, gefitinib,erlotinib, panitumumab, PKI-166, EKB-569, HKI-272 (WAY-177820),icotinib, brigatinib, afatinib, lapatinib, canertinib, AEE788, XL647,and Zactima. The EGFR-targeted agent is more preferably cetuximab,gefitinib, erlotinib, or panitumumab, and most preferably cetuximab.

Unless stated otherwise in this specification, the expression “measuringthe expression level of the gene; or the expression or activity level ofthe protein of the gene” used in this specification means detecting atarget to be detected in the corresponding sample.

In the present disclosure, the target to be detected is mRNA and/orprotein of the corresponding gene in the sample. That is, whether thegene is expressed or not may be verified by detecting RNA as atranscription product of the gene or protein (preferably, the activeform) as a gene product.

The detecting of the RNA or the protein may be generally implemented byextracting the RNA or the protein from the sample to detect the RNA orthe protein from the extractant. The detecting of the RNA or the proteinmay be measured by an immunological analytical method, a hybridizationreaction, and an amplification reaction, but is not limited thereto andmay be easily implemented by using various techniques known in the art.

Further, according to a preferred embodiment of the present disclosure,the agent that measures the gene expression level includes antisenseoligonucleotide, a primer pair, or a probe which is specifically boundto the mRNA of the gene.

The agent that measures the expression of the mRNA is selected from thegroup consisting of antisense oligonucleotide, a primer pair, a probe,and a combination thereof that are specific to the gene. That is, thedetecting of nucleic acid may be performed by an amplification reactionusing one or more oligonucleotide primers which are hybridized to anucleic acid molecule encoding the gene or a complementary material ofthe nucleic acid molecule.

For example, the detecting of the mRNA using the primer may be performedby verifying whether the gene is amplified by a known method in the artafter amplifying a gene sequence by using the amplification method suchas PCR.

Further, according to a preferred embodiment of the present disclosure,the agent that measures the expression or activity level of the proteinincludes an antibody, a peptide, or a nucleotide which is specificallybound to the protein.

The agent that measures the expression or activity level of the proteinmeans an antibody which is specifically bound to the protein andincludes a polyclonal antibody, a monoclonal antibody, a recombinantantibody, and a combination thereof.

The antibody includes all of not only the polyclonal antibody, themonoclonal antibody, the recombinant antibody, and a complete formhaving two light chains with the full length and two heavy chains withthe full length, but also functional fragments of the antibodymolecules, for example, Fab, F(ab'), F(ab')2, and Fv. The antibodies maybe easily prepared by using a well-known technique in the art andantibodies which are prepared and commercially sold may be used.

The composition of the present disclosure may further include labelswhich may quantitatively or qualitatively measure formation of anantigen-antibody complex, general tools used in the immunologicalanalysis, reagents, and the like as well as the agent for measuringwhether the above-described gene is expressed or not.

The labels which may quantitatively or qualitatively measure theformation of the antigen-antibody complex include enzymes, fluorescentsubstances, ligands, luminescent substances, microparticles, redoxmolecules, radioactive isotopes, and the like, and are not necessarilylimited thereto. The enzymes usable as the detection label includeβ-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, peroxidase,alkaline phosphatase, acetylcholinesterase, glucose oxidase, hexokinaseand GDPase, RNase, glucose oxidase and luciferase, phosphofructokinase,phosphoenolpyruvate carboxylase, aspartate aminotransferase,phosphenolpyruvate decarboxylase, β-lactamase, and the like, and are notlimited thereto. The fluorescent substances include fluorescein,isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin,o-phthaldehyde, fluorescamine, and the like, and are not limitedthereto. The ligands include biotin derivatives and the like, and arenot limited thereto. The luminescent substances include acridiniumester, luciferin, luciferase, and the like, and are not limited thereto.The microparticles include colloidal gold, colored latex, and the like,and are not limited thereto. The redox molecules include ferrocene,ruthenium complex compounds, viologen, quinone, Ti ions, Cs ions,diimide, 1,4-benzoquinone, hydroquinone, K₄W(CN)⁸, [Os(bpy)₃]²⁺,[RU(bpy)₃]²⁺, [MO(CN)₈]⁴⁻, and the like, and are not limited thereto.The radioactive isotopes include ³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co,⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ₁₂₅I, ¹³1I, ¹⁸⁶Re, and the like, and are not limitedthereto.

An example of the tool or the reagent includes suitable carriers,solubilizing agents, detergents, buffering agents, stabilizers, and thelike, but is not limited thereto. When the marker is the enzyme, asubstance and a quencher which may measure the enzyme activity may beincluded. The carriers include a soluble carrier, and an insolublecarrier. An example of the soluble carrier includes a buffer solutionthat is physiologically acceptable and known in the art, for example,PBS, and an example of the insoluble carrier may include polystyrene,polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesin,cross-linked dextran, polysaccharide, and other papers, glass, metal,agarose, and a combination thereof.

Since the composition of the present disclosure uses the aforementionedbiomarker, the disclosure of the duplicated contents is omitted foravoiding excessive complexity of this specification.

In yet another aspect of the present invention, there is provided a kitfor predicting the susceptibility to the EGFR-targeted agent includingthe composition.

The kit may include a tool, a reagent, and the like which are generallyused in the art for immunological analysis as well as the agent whichmeasures the expression of the gene; or the expression or activity levelof the protein thereof.

An example of the tool or the reagent includes suitable carriers,markers capable of generating a detectable signal, chromophores,solubilizing agents, detergents, buffering agents, stabilizers, and thelike, but is not limited thereto. When the marker is the enzyme, asubstance and a quencher which may measure the enzyme activity may beincluded. The carriers include a soluble carrier, and an insolublecarrier. An example of the soluble carrier includes a buffer solutionthat is physiologically acceptable and known in the art, for example,PBS, and an example of the insoluble carrier may include polystyrene,polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesin,cross-linked dextran, polysaccharide, polymers such as magneticparticles plating a metal on the latex, and other papers, glass, metal,agarose, and a combination thereof.

Since the kit of the present disclosure uses the aforementionedbiomarker and composition as the configuration, the disclosure of theduplicated contents is omitted for avoiding excessive complexity of thisspecification.

In still another aspect of the present invention, there is provided asusceptibility enhancer or a composition for enhancing susceptibility toan epidermal growth factor receptor (EGFR)-targeted agent for a subject,including an inhibitor of expression of an RON (Recepteur d' OrigineNantais, NM_002447.1) gene; or expression or activity of the protein ofthe gene as an active ingredient.

According to a preferred embodiment of the present disclosure, when theKRAS (V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog,NM_033360.2), NRAS (neuroblastoma RAS viral (v-ras) oncogene homolog,NP_002515.1), or BRAF (v-raf murine sarcoma viral oncogene homolog B,NP_004324.2) gene is a wild type, the target predicts the susceptibilityto the EGFR-targeted agent and may be applied to a case where cancercells with KRAS, NRAS, or BRAF mutation do not achieve a desired effect.

According to the present disclosure, in cancer cells with a KRAS, NRAS,or BRAF wild-type gene in which activity of the RON gene and an activeprotein as a product thereof are expressed, a result exhibits that adeath rate of the cancer cells by cetuximab is significantly decreasedas compared with anticancer cells in which the activity of the RON geneand the active protein are not expressed.

Further, in the present disclosure, in the cancer cells with a KRAS,NRAS, or BRAF wild-type gene in which the activity of the RON gene andthe active protein as a product thereof are expressed, when theexpression of the RON gene; or the expression or activity of the proteinis inhibited, a result exhibits that a death rate of the cancer cells bycetuximab is significantly increased.

Accordingly, this indicates that the susceptibility of the cancer cellsto cetuximab is enhanced by the presence of the KRAS, NRAS, or BRAFwild-type (normal) gene (normal expression and function of the normalgene); and the expression inhibition of the RON gene, and the presentdisclosure provides an excellent effect of enhancing the susceptibilityof the target to the EGFR-targeted agent based thereon.

The susceptibility enhancer or the composition for enhancing of thepresent disclosure may additionally include a carrier which ispharmaceutically acceptable. The pharmaceutically acceptable carrierwhich may be used in the present disclosure may be used by selectinggeneral excipients, disintegrants, binders, lubricants, and otheradditives, for example, stabilizers, relaxant, emulsifiers, and thelike. For example, as the excipients, microcrystalline cellulose,lactose, low-substituted hydroxypropyl cellulose, and the like may beused, and as the disintegrants, sodium starch glycolate, anhydrousdibasic calcium phosphate, and the like may be used. As the binders,polyvinylpyrrolidone, low-substituted hydroxypropyl cellulose, and thelike may be used, and the lubricants may be selected and used frommagnesium stearate, silicon dioxide, talc, and the like.

In the present disclosure, the presence of the KRAS, NRAS, or BRAFwild-type gene and the expression inhibition of the RON gene reduce thegrowth of the cancer cells in the treatment of the EGFR-targeted agent.

The expression of the RON gene is inhibited by one or more selected fromthe group consisting of small interference RNA (siRNA), short hairpinRNA (shRNA), microRNA (miRNA), ribozyme, DNAzyme, peptide nucleic acids(PNAs), antisense oligonucleotide, antibodies, aptamers, naturalextracts, and chemical substances that are specifically bound to mRNA ofthe RON gene.

The expression of the RON gene is inhibited by more preferably antisenseoligonucleotide, aptamers, siRNA, shRNA, or miRNA, and most preferablysiRNA or antisense oligonucleotide.

The term “siRNA” used in this specification means a small RNA fragmentwith a size of 21 to 25 nucleotides generated when a double-stranded RNAis cleaved by a dicer and inhibits the expression by specifically boundto mRNA having a complementary sequence. On the purpose of the presentdisclosure, the term “siRNA” means inhibiting the expression of the geneby specifically bound to RON mRNA. The siRNA may be chemically orenzymatically synthesized. A preparation method of the siRNA is notparticularly limited and may use a known method in the art.

According to the preferred embodiment of the present disclosure, thesiRNA includes a nucleotide sequence of SEQ ID NO: 13.

The term “antisense oligonucleotide” used in this specification is anucleotide sequence that inhibits the expression by complementarilybound to the miRNA and is not limited thereto, but includes antisenseRNA, antisense DNA, and antagonist mRNA.

Since the susceptibility to the EGFR-targeted agent is enhanced by usingthe expression level of the aforementioned biomarker in the method ofthe present disclosure, the disclosure of the duplicated contents isomitted for avoiding excessive complexity of this specification.

In still yet another aspect of the present invention, there is provideda pharmaceutical composition for preventing or treating diseases relatedwith dysregulation of an EGFR signaling pathway, including asusceptibility enhancer to the aforementioned EGFR-targeted agent andthe EGFR-targeted agent as the active ingredients.

In the present disclosure, the diseases related with the dysregulationof the EGFR signaling pathway are cancer, atherosclerosis, pulmonaryfibrosis, renal fibrosis and regeneration, liver disease, allergicdisease, inflammatory disease, autoimmune disorder, cerebrovasculardisease, cardiovascular disease, or symptoms associated with organtransplantation, and preferably cancer.

That is, the inhibitor of the expression of the RON (Recepteur d'Origine Nantais, NM_002447.1) gene as the susceptibility enhancer to theEGFR-targeted agent of the present disclosure; or the expression oractivity of the protein of the gene increases the susceptibility to theanticancer and increases the anticancer effects of the anticancer agentswhen being administrated together with the anticancer agents to morefacilitate the treatment of the cancer.

The “cancer” which is the disease to be improved, prevented, or treatedby the composition of the present disclosure means a common name ofdiseases caused by cells having an aggressive characteristic in whichthe cells are divided and grown regardless of a normal growth limit, aninvasive characteristic in which the cells are invaded to the ambienttissue, and a metastatic characteristic in which the cells spread toother parts of the body. In this specification, the cancer is used asthe same meaning as malignant tumor.

The cancer to which the composition of the present disclosure is appliedincludes breast cancer, lung cancer, stomach cancer, liver cancer, bloodcancer, bone cancer, pancreatic cancer, skin cancer, head or neckcancer, cutaneous or intraocular melanoma, uterine sarcoma, ovariancancer, rectal cancer, anal cancer, colon cancer, fallopian tubecarcinoma, endometrial carcinoma, cervical cancer, small intestinecancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenalcancer, soft tissue tumor, urethral cancer, prostate cancer,bronchogenic cancer, bone marrow tumor, and the like, but is not limitedthereto.

Preferably, the composition of the present disclosure may be applied toprevent or treat the colon cancer.

The term “prevention” in this specification means that it has been notdiagnosed that the diseases or the disorders are preserved, butgeneration of the diseases or the disorders is suppressed in animalswhich are susceptible to the diseases or the disorders. The term“treatment” in this specification means (i) suppression of developmentof the diseases or the disorders; (ii) reduction of the diseases or thedisorders; and (iii) removal of the diseases or the disorders.

Further, the composition of the present disclosure may further include apharmaceutically acceptable carrier.

The pharmaceutically acceptable carrier included in the pharmaceuticalcomposition of the present disclosure is generally used in thepreparation, and includes lactose, dextrose, sucrose, sorbitol,mannitol, starches, gum acacia, calcium phosphate, alginate, gelatin,calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone,cellulose, water, syrup, methyl cellulose, methylhydroxy benzoate,propyl hydroxybenzoate, talc, magnesium stearate, mineral oils, and thelike, but is not limited thereto. The pharmaceutical composition of thepresent disclosure may additionally include a lubricant, a wettingagent, a sweetener, a flavoring agent, an emulsifier, a suspension, apreservative, and the like other than the components. The suitablepharmaceutically acceptable carrier and agent are disclosed in detail inRemington's Pharmaceutical Sciences (19th ed., 1995).

A suitable dosage of the pharmaceutical composition of the presentdisclosure may be prescribed by various methods according to factors,such as formulation methods, administration methods, patient's age, bodyweight, sex, morbidity, food, time of administration, a route ofadministration, an excretion rate, and response susceptibility.

Meanwhile, the dosage of the pharmaceutical composition of the presentdisclosure is preferably 0.001 to 1000 mg/kg (weight) per day.

The pharmaceutical composition of the present disclosure may be orallyor parenterally administrated, and in the case of the parentaladministration, the pharmaceutical composition may be administrated byIntravenous injection, subcutaneous injection, intramuscular injection,intraperitoneal injection, dermal administration, and the like. It ispreferred that the administration route is determined according to akind of disease to which the pharmaceutical composition of the presentdisclosure is applied.

The concentration of the corresponding gene or the expression inhibitorof the protein thereof in the enhancer as the active ingredient includedin the composition of the present disclosure is determined byconsidering a therapeutic purpose, a patient's condition, a requiredperiod, the severity of the disease, and the like and is not limited toa concentration in a predetermined range.

The pharmaceutical composition of the present disclosure is formulatedby using the pharmaceutically acceptable carrier and/or the excipientaccording to a method which may be easily implemented by those skilledin the art to be prepared in a unit capacity form or inserted into alarge-capacity container to be prepared. In this case, the formulationmay be a solution in oil or an aqueous medium, a suspension or anemulsion, or an X agent, a powder, a granule, a tablet or a capsule, andmay additionally include a dispersing agent or a stabilizing agent.

Since the composition of the present disclosure improves the cell deathof the cancer cells by using the EGFR-targeted agent as theaforementioned susceptibility enhancer and the anticancer agent, thedisclosure of the duplicated contents is omitted for avoiding theexcessive complexity of this specification.

In still yet another aspect of the present invention, there is provideda method of predicting susceptibility to an EGFR-targeted agentincluding:

(a) preparing a biological sample from a subject;

(b) measuring an expression level of a RON (Recepteur d' OrigineNantais, NM_002447.1) gene; or an expression or activity level of aprotein of the gene in the biological sample; and

(c) determining susceptibility to the EGFR-targeted agent of the subjectbased on the verified result of the level measured in step (b).

According to the preferred embodiment of the present disclosure, thetarget has a wild-type gene such as KRAS (V-Ki-ras2 Kirsten rat sarcomaviral oncogene homolog, NM_033360.2), NRAS (neuroblastoma RAS viral(v-ras) oncogene homolog, NP_002515.1), or BRAF (v-raf murine sarcomaviral oncogene homolog B, NP_004324.2).

The prediction method of the present disclosure is constituted byincluding processes of determining that the corresponding sample hassusceptibility to the EGFR-targeted agent when the expression or theactivity of the disclosed gene or protein is inhibited or reduced ascompared with a normal sample after obtaining a biological sample from atarget patient and measuring whether one gene or a plurality of genesselected from the group consisting of the aforementioned genes in thesample is expressed; and determining that the corresponding sample hasresistance to the EGFR-targeted agent when the expression or theactivity of the disclosed gene or protein is increased or enhanced.

The prediction method of the present disclosure characterizes thatwhether a specific gene or protein (preferably, an active form) isexpressed in the sample is an index of the susceptibility to theanticancer agent of the cancer cells.

According to the preferred embodiment of the present disclosure, in step(b), an expression level of any one gene selected from the groupconsisting of EGFR (Epidermal Growth Factor Receptor, Gene Bankaccession No. U48722.1), Adam11 (ADAM metallopeptidase domain 11,NM_002390.4), Adam32 (ADAM metallopeptidase domain 32, NM_145004.5),FZD4 (Frizzled family receptor 4, NM_012193.2), GPER (G protein-coupledestrogen receptor 1, NM_001505.2), and GPR101 (G protein-coupledreceptor 101, NM_054021.1) genes and a combination thereof; anexpression or activity level of the protein of the gene is additionallymeasured.

According to the preferred embodiment of the present disclosure, in step(c), when the expression level of the RON gene; or the expression oractivity level of the protein thereof is low as compared with the normallevel, it is determined that there is the susceptibility(susceptibility) to the EGFR-targeted agent of the target.

According to the preferred embodiment of the present disclosure, in step(c), when the expression level of the RON gene; or the expression oractivity level of the protein thereof is high as compared with thenormal level, it is determined that there is the resistance to theEGFR-targeted agent of the target.

According to the preferred embodiment of the present disclosure, whenthe expression level of any one gene selected from the group consistingof the Adam11, Adam32, FZD4, GPER, GPR101 genes and a combinationthereof; an expression or activity level of the protein of the gene islow as compared with a normal level, it is determined that there is thesusceptibility (susceptibility) to the EGFR-targeted agent of thetarget.

More particularly, in step (c) of the present disclosure, based on theresult of the expression level measured in step (b), when it is verifiedthat the KRAS, NRAS, or BRAF wild-type gene is included and the level ofthe activity of the RON gene and the active protein which is the productthereof is inhibited and/or reduced as compared with a normal(wild-type) value, it is determined that the corresponding tumor cellsobtained from the target patient has the susceptibility to theEGFR-targeted agent as the anticancer agent. In this case, additionally,when the expression level of any one gene selected from the groupconsisting of the Adam11, Adam32, FZD4, GPER, and GPR101 genes and acombination thereof; the expression or activity level of the protein ofthe gene is measured and it is determined that the level is inhibitedand/or reduced as compared with a normal (wild-type) value, it isdetermined that the corresponding tumor cells obtained from the targetpatient has the susceptibility(susceptibility) to the EGFR-targetedagent as the anticancer agent.

In this specification, the term “low expression” or “low activity” usedwhile mentioning the expression level of the genes is referred to as avalue of the biomarker detected in the biological sample obtained from ahealthy or normal target or an target as a comparison target or a valueor level of the biomarker in the biological sample which is lower than alevel range, when the biomarker represents abnormal process, disease, orother conditions in the target or a symptom thereof. Further, the term“low expression” or “low activity” may be referred to as a “differentiallevel”, “differential value”, or “differently expressed” as comparedwith the “normal” expression level or value of the biomarker, andincludes both a quantitative difference and a qualitative difference inthe expression.

According to the preferred embodiment of the present disclosure, themeasuring of the expression level of the RON gene; or the expression oractivity level of the protein of the gene additionally includesmeasuring whether a splicing variant or a mutant of the gene is present.

According to the present disclosure, the presence of the variant of thegene or one or more mutants in the gene causes a change of theexpression aspect of the gene to have an effect on the susceptibility tothe EGFR-targeted agent (see Example 3).

The term “variant” used in this specification means RON isoformgenerated when an exon site of the corresponding gene is deleted byalternative splicing, and an activity degree of the RON is adjusted byalternative splicing which is one of important processes of the geneexpression regulation in eukaryotes.

RONΔ155 (alternatively, RON Delta 155), RONΔ160 (alternatively, RONDelta 160), and RONΔ165 (alternatively, RON Delta 165) used in thepresent disclosure are generated by skipping of the exons through thesplicing and always structurally activated without ligands.

According to the present disclosure, splicing variants in which theexons are deleted by an alternative splicing mechanism of the RON havedifferent sensitivities to the drug according to the expression thereof.

The term “mutant” or “variant” used in this specification includes basesubstitution, deletion, insertion, amplification, and rearrangement ofthe nucleotide and the amino acid sequence of the corresponding gene.The nucleotide variant is referred to as a change (for example,insertion, deletion, inversion or substitution of one or morenucleotides, for example, a single nucleotide polymorphism (SNP)), inthe nucleotide sequence for a reference sequence (for example, awild-type sequence). The term includes a corresponding change of acomplement of the nucleotide sequence unless exhibited otherwise. Thenucleotide variant may be somatic mutation or pattern polymorphism.

Further, the amino acid variant is referred to as a change (for example,insertion, substitution, or deletion one or more amino acids, forexample, internal deletion or truncation of N- or C-terminal) of theamino acid sequence as compared with the reference sequence (forexample, the wild-type sequence).

In the embodiment of the present disclosure, the mutant is E387A andH424L point mutants in which one amino acid is substituted.

Further, it was verified that the E387 site and the H424 site of theamino acid of the RON protein are sites capable of bound to the EGFRprotein.

The detection of the variant may be performed by targeted moleculecloning and sequence analysis by using a well known technique in theart. For example, DNA sequence analysis; primer extending assayincluding allele-specific nucleotide mixing assay and allele-specificprimer extending assay (for example, allele-specific PCR,allele-specific ligation chain reaction (LCR), and gap-LCR);allele-specific oligonucleotide hybridizing assay (for example,oligonucleotide ligation assay); a cleave protection assay that detectsmismatched nucleotides in a double strand of nucleic acid by usingprotection from a cleaver; MutS protein binding analysis;electrophoresis analysis comparing mobility of a variant and a wild typenucleic acid molecule; deformation-gradient gel electrophoresis (DGGE,for example, the same as the literature [Myers et al, (1985) Nature313:495]); analysis of RNase cleavage in mismatched nucleotide pairs;analysis of chemical or enzymatic cleavage of a hetero double-strandedDNA; mass spectrometry (for example, MALDI TOF); genetic bit analysis(GBA); 5′ nucleases assay (for example, TaqMan); and an assay usingmolecular beacon are included, but the methods are not limited thereto.

The term “biological sample” used in this specification means allsamples obtained from the target in which the expression of thebiomarker of the present disclosure may be detected.

According to the preferred embodiment of the present disclosure, thebiological sample is any one selected from the group consisting ofsaliva, biopsy, blood, skin tissue, liquid culture, feces, and urine andis not particularly limited thereto, and may be treated and prepared bya method which is generally used in the art.

Since the method of the present disclosure determines that there is thesusceptibility by using the aforementioned biomarker, the disclosure ofthe duplicated contents is omitted for avoiding excessive complexity ofthis specification.

In yet another aspect of the present invention, there is provided amethod of enhancing susceptibility to the EGFR-targeted agent including:administering both of a susceptibility enhancer to the aforementionedEGFR-targeted agent and the EGFR-targeted agent to the target.

Since the method of the present disclosure enhances the susceptibilityby using the aforementioned susceptibility enhancer and theEGFR-targeted agent as the anticancer agent, the disclosure of theduplicated contents is omitted for avoiding the excessive complexity ofthis specification.

Hereinafter, these Examples are only for describing the presentdisclosure in more detail, and it will be apparent to those skilled inthe art that the scope of the present disclosure is not limited to theseExamples according to the gist of the present disclosure.

Experimental Method and Condition

Immunoprecipitation Method

In order to analyze a RON active form in a colon cancer cell line, after500 μg of a lysate of the colon cancer cell line was mixed with 1 μg ofan anti-RON antibody, cultured for 12 hours at 4° C., added with 20 μlof a protein-sepharose bead (Santa Cruz Biotehcnology, Santa Cruz,Calif., USA), and then additionally reacted for 2 hours. Theimmunoprecipitate was washed with a buffer solution (Nondiet P-40 lysisbuffer) five times, added and heated with 20 μl of a 2XSDS samplesolution, and then western blotting was performed by using anti-RON(Santa Cruz Biotechonology) and anti-phospho-Tyrosine (CellSignaling,Beverly, Calif., USA) antibodies.

In order to analyze interaction of endogenous RON and EGFR, 300 μg of alysate of a HCT-8 cell line was mixed with 1 μg of an anti-RON antibodyor 1 μg of an anti-rabbit IgG antibody, cultured for 12 hours at 4° C.,added with 20 μl of the protein-sepharose bead (Santa CruzBiotehcnology, Santa Cruz, Calif., USA), and then additionally reactedfor 2 hours. The immunoprecipitate was washed with a buffer solution(Nondiet P-40 lysis buffer) five times, added and heated with 20 μl of a2XSDS sample solution, and then western blotting was performed by usinganti-RON (Santa Cruz Biotechonology) and anti-EGFR(Cell Signaling,Beverly, Calif., USA) antibodies. In order to analyze interaction ofexogenous RON and EGFR, a Δ160 variant as a RON active form wastransfected in a LoVo colon cancer cell line without expressing the RONprotein itself, a cell lysate was collected after 48 hours, 300 μg ofthe cell lysate was mixed with 1 μg of an anti-RON antibody or 1 μg ofan anti-rabbit IgG antibody, cultured for 12 hours at 4° C., added with20 μl of the protein-sepharose bead (Santa Cruz Biotehcnology, SantaCruz, Calif., USA), and then additionally reacted for 2 hours. Theimmunoprecipitate was washed with a buffer solution (Nondiet P-40 lysisbuffer) five times, added and heated with 20 μl of a 2XSDS samplesolution, and then western blotting was performed by using anti-RON(Santa Cruz Biotechonology) and anti-EGFR (Cell Signaling, Beverly,Calif., USA) antibodies.

Microarray Analysis

A Colo320HSR colon cancer cell line without expressing the RON proteinwas transfected for 48 hours with a construct (plasmid) expressing RONactive form Δ160 and/or c-MET. Thereafter, the lysate of the cells wasanalyzed by a microarray.

*Gene Overexpression and Suppression Methods

In order to analyze interaction of exogenous RON and EGFR, a constructexpressing Δ160 as a RON active form was transfected for 48 hours into aLoVo colon cancer cell line in which the RON protein was non-expressed.

In order to analyze the endogenous interaction of RON and EGFR, RONsiRNA(small interfering RNA) was transfected for 48 hours in the HCT-8colon cancer cell line in which RON was activated.

The RON siRNA sequence (SEQ ID NO: 13) is as follows:5′-ACUUUGUAGAGGAGUUUGAUU-3′.

Reverse Transcription (RT)-PCR and Real-Time PCR

In order to perform the RT-PCR, Colo320HSR colon cancer cell line inwhich the RON was non-expressed was transfected with constructexpressing the RONΔ160 as the RON active type form for 48 hours andKM12C colon cancer cell line in which the RON was activated wastransfected with the RON siRNA for 48 hours. The RNA was extracted byusing trizol (Cat.#15596-026, Life technologies™), respectively. Theextracted RNA was synthesized to cDNA by using a RT-PCR Kit (AccuPowerRT PreMix, Bioneer). The difference in expression of the correspondinggene was verified by PCR (AccuPower PCRPreMix, Bioneer) by using thesynthesized cDNA and a gene-specific primer and quantified by using areal-time PCR (LightCycler 480 SYBR Green, Roche).

TABLE 11 Primer sequences Genes Forward Reverse Adam11 5′- 5′-TGGCTTCCTCCTCTGTG GCACTTCCCTTCATTG TCAA-3′ CTGC-3′ (SEQ ID NO: 1)(SEQ ID NO: 2) Adam32 5′- 5′- AATGGCAGATTGGAGG TTCATAGCAGGCAAATGAAATG-3′ GGAGCA-3′ (SEQ ID NO: 3) (SEQ ID NO: 4) FZD4 5′- 5′-TGACTGGCTTGTGCTA ATGCCTGAAGTGATGC TGTTGG-3′ CCAC-3′ (SEQ ID NO: 5)(SEQ ID NO: 6) GPR101 5′- 5′- GGCAGAATGGAAGCC TTGCTGTTACGACGAC AAGGA-3′TGGGTG-3′ (SEQ ID NO: 7) (SEQ ID NO: 8) GPER 5′- 5′- ATCGTGCCCTTCGCCACCAGTCGTGAGGTTTC TCAT-3′ CTAAGCAG-3′ (SEQ ID NO: 9) (SEQ ID NO: 10)GAPDH 5′- 5′-AGG GGC CAT CCA AGAAGGCTGGGGCTCA CAG TCT TC-3′ TTTG-3′(SEQ ID NO: 12) (SEQ ID NO: 11)

TABLE 2 Reverse transcription - PCR (polymerase chain reaction)condition 42° C. 90° C. 60 min 5 min

TABLE 3 PCR condition 1 cycle 35 cycle 1 cycle 95° C. 95° C. 60° C. 72°C. 72° C. 5 min 30 sec 1 min 1 min 10 min

TABLE 4 Quantitative real-time PCR condition 1 cycle 50 cycle 1 cycle95° C. 95° C. 58° C. 72° C. 72° C. 5 min 30 sec 30 sec 1 min 10 min

Western Blot

In order to perform the western blot, a protein separated from each cellwas separated through SDS-PAGE and transferred to polyscreen membranes(New England Nuclear, Boston, Mass., USA), reacted for 12 hours at 4° C.by using various antibodies (anti-phospho RON (MyBioSource, San Diego,Calif., USA), anti-phospho Tyrosine, anti-EGFR, anti-phospho EGFR (Cellsignaling Technology, Beverly, Mass., USA), anti-RON, and anti-r-tubulin(Santa Cruz Biotechnology), and then washed with a 1XTBS-T buffersolution for 10 minutes three times. A suitable anti-rabbit-HRP oranti-mouse-HRP secondary antibody reacted for 2 hours at roomtemperature and was washed with the 1XTBS-T buffer solution for 10minutes three times to verify the expression of the protein by using anECL solution (Amersham, Buckinghamshire, UK).

Phospho-RTK Array

After the Δ160 construct as the RON active form was transfected for 48hours in the Colo320HSR cell line without expressing the RON protein,the cell lysate was collected. 50 μg of the collected cell lysate wasreacted with a membrane included in a human phospho-RTK array kit (R&DSystems, Inc, Minneapolis, Minn., USA) for 12 hours at 4° C., washedwith a TBS-T buffer solution three times, reacted with ananti-phospho-Tyrosine HRP antibody for 2 hours at room temperature, andthen washed with the TBS-T buffer solution three times. Thereafter, inthe membrane, a change in the expression of receptor tyrosine kinase(RTK) proteins was verified by using the ECL solution (Amersham,Buckinghamshire, UK).

Treatment of Anticancer Agent

After 5, 10, and 20 μg/ml of cetuximab (Merck & Co., Inc, N.J., USA)were treated for 48 hours in CaCo2 and LIM1215 colon cancer cell linesin which the RON was not activated and a KM12C cell line in which theRON was activated, a cell solution was collected to perform trypan bluecounting (FIG. 3A).

KM12C colon cancer cell line in which the RON was activated was treatedwith RON siRNA for 48 hours, was treated with 20 μg/ml of cetuximab for48 hours, and then the cell solution was collected to perform trypanblue counting.

Treatment of Inhibitor

3 μm of an RON inhibitor (LY2801653) and 20 μg/ml of an EGFR-targetedagent (cetuximab) were treated for 48 hours in the KM12C colon cancercell line in which the RON was activated. After treatment, the celllysate was collected to perform trypan blue counting.

Further, narnatumab (Creative Diagnostic#TAB-184 Anti-Human RONTherapeutic Antibody) as an RON-target antibody anticancer agent and 100μg/ml of an EGFR-targeted agent (cetuximab) were treated for 48 hours.After treatment, the cell death induction degree was verified by thesame method as above.

Mutagenesis of Point Mutant

The mutagenesis was performed according to a guideline provided from thekit by using a muta-Direct mutagenesis kit (Intron, Cat No. 15071).Primer sequences used in mutagenesis are as follows.

RON (E387A); Forward: 5′-GGA GCG CTG TTG TGC ATC CCC AGT CCA TCC-3′,Reverse: 5′-GGA TGG ACT GGG GAT GCA CAA CAG CGC TCC-3′, RON(H424L);Forward: 5′-ACA CCA GCT GCC GCC TCT TCC CTC TGC TGG-3′, Reverse:5′-CCA GCA GAG GGA AGA GGC GGC AGC TGG TGT-3′, RON(K1114M); Forward:5′-AAT GTG CCA TCA TGT CAC TAA GTC G-3′, Reverse:5′-CGA CTT AGT GAC ATG ATG GCA CAT T-3′, EGFR (D813N); Forward:5′-TTG GTG CAC CGC AAC CTG GCA GCC AGG-3′, Reverse:5′-CCT GGC TGCCAG GTT GCG GTG CAC CAA-3′.

Mutation was progressed by using the primer and a muta-direct enzyme andthen transformation was progressed in E. coli. After DNA was extractedfrom colonies, mutation was verified through a sequencing analysismethod.

RON Activity Analysis in Colon Cancer Cell Line or Colon CancerPatient's Tissue

In order to analyze the RON activity, after a protein was extracted froma cell line or a colon cancer patient's tissue (by using a RIPA buffer),activity was verified by using a phospho-RON antibody (Mybiosource,MBS462024, dilution factor 1:1000) in 20 μg of the cells and 50 μg ofthe colon cancer patient's tissue.

Further, after immunoprecipitation was performed with the RON antibody(santa cruz; sc-322, Lot#G1514, 2 μg, incubation of 2 days) (by using300 μg of the cell line and the colon cancer patient's tissue),phosphorylation was verified by a phospho-tyrosine antibody (Cellsignaling; 9411, dilution factor 1:1000). In order to verify RONΔ155 andRONΔ160 (alternative splicing form) which were the RON active forms, RNAwas extracted from the colon cancer cell line or the colon cancerpatient's tissue with a trizol reagent.

After analyzing the cDNA, analysis was performed by using two primers.

First, a primer designed for distinguishing RONΔ155 and RONΔ160 was usedand the primer was as follows: Forward: 5′-CTCTGGGGACCAGGTTTTCC-3′,Reverse: 5′-ACCATCAATGGCAGGGAGTG-3′. In the RT-PCR condition, 37 cycleswere performed for 5 minutes at 94° C., for 30 seconds at 94° C., for 30seconds at 63° C., and for 1 minute 30 seconds at 72° C. and extendedfor 10 minutes at 72° C. RON wild type was verified 1552 bp, RONΔ155 wasverified 1078 bp, and RONΔ160 was verified 1225 bp.

In order to further verify the RT-PCR condition, the primers reported inan existing literature were used and the primers were as follows.Forward: 5′-TGG TCA GTA GCA GCT TCT CA-3′, Reverse: 5′-AGG CAG CAG GATACC AAG GA-3′. In the RT-PCR condition, 38 cycles were performed for 5minutes at 94° C., for 30 seconds at 94° C., for 45 seconds at 57° C.,and for 1 minute at 72° C. and extended for 10 minutes at 72° C. RONwild type was verified 1.6 kb, RONΔ160 was verified 1.3 kb, and RONΔ155was verified 1.1 kb.

Example 1. Activity of EGFR According to Activity of RON Protein

1-1. Presence of RON Protein Active form in Human Colon Cancer Cell Line

The present inventors selected a colon cancer cell line in which the RONprotein was activated or de-activated and verified the RONphosphorylation in a total of 19 human colon cancer cell lines by usingwestern blotting.

The phosphorylated RON protein was regarded as the RON active formprotein.

As a result, as illustrated in FIG. 1A, in four cell lines HT-29, KM12C,KM12L4, and SW1417 among 18 cell lines, the expression of thephosphorylated RON protein was observed and it can be seen that the fourcell lines had the RON activity.

Meanwhile, in the LoVo and Colo320HSR colon cancer cell lines, it wasexhibited that the RON protein was absent.

1-2. Analysis of Gene Expression according to Activation of RON Proteinusing DNA Microarray Analysis

In order to analyze the gene expression level according to the RONexpression in the Colo320HSR cell line in which the RON protein wasabsent, the present inventors simultaneously overexpressed the Δ160variant which was active form of both MET and RON in the Colo320HSR celland then performed the microarray.

The overexpression of the RON gene was considered as the activation ofthe RON gene or protein.

As illustrated in FIG. 1B, the genes changed by only the RON activationwere analyzed by comparing with genes in a sample group which wereoverexpressed only the MET gene, and as a result, five genes Adam11,Adam32, FZD4, GPER, and GPR101 associated with the EGFR were identifiedas the genes changed by the RON.

1-3. Real-Time Polymerase Chain Reaction (PCR) Analysis of EGFRTransactivation-Related Genes according to RON Activation in ColonCancer Cell Line

In order to analyze the expression level change of the EGFRtransactivation-related genes according to the RON activation, thepresent inventors performed the real-time PCR with respect to the fivegenes (Adam11, Adam32, FZD4, GPER, and GPR101) associated with the EGFRtransactivation which was estimated that the expression was changed bythe RON, after the Δ160 variant as the RON activation form wasoverexpressed in the Colo320HSR colon cancer cell line in which the RONwas not expressed.

As a result, as illustrated in FIG. 1C, it was observed that theexpression of the Adam11, Adam32, FZD4, GPER, and GPR101 genes wassignificantly increased by RON overexpression.

1-4. RT-PCR (Reverse Transcription Polymerase Chain Reaction) Analysisof EGFR Transactivation-Related Genes according to RON Activation inColon Cancer Cell Line

In order to analyze the expression level change of the EGFRtransactivation-related genes according to the RON activation, theinventors performed the PCR with respect to the five genes Adam11,Adam32, FZD4, GPER, and GPR101 associated with the EGFR transactivationwhich was estimated that the expression was changed by the RON, afterthe Δ160 variant as the RON activation form of the RON gene wasoverexpressed in the Colo320HSR colon cancer cell line in which the RONwas not expressed.

As a result, as illustrated in FIG. 1D, by the RON activation, it wasobserved that the expression of the Adam11, Adam32, FZD4, GPER, andGPR101 genes was significantly increased in the sample group in whichthe Δ160 variant as the RON activation form of the RON gene wasoverexpressed.

1-5. Real-Time PCR Analysis of EGFR Transactivation-Related Genes inColon Cancer Cell Line in which RON is Inhibited

The inventors analyzed the expression of the genes Adam11, Adam32, FZD4,GPER, and GPR101 associated with the EGFR transactivation with real-timePCR after artificially suppressing the RON activity by using an siRNAtechnique in the KM12C colon cancer cell line in which the RON wasactivated, in order to analyze the change aspect of the expression ofthe EGFR transactivation-related genes according to the RON inhibition.

As a result, as illustrated in FIG. 1E, it was exhibited that the FZD4and GPR101 genes were significantly reduced by inhibition of the RONactivation.

1-6. Analysis of Activity of EGFR Protein by RON Activation

As described above, it was verified that the EGFRtransactivation-related genes which were identified in Example 1-2 (FIG.1B) and verified in Examples 1-3 to 1-5 (FIGS. 1C to 1E) was expressedby the RON activation. Accordingly, the inventors verified whether thephosphorylated protein of the EGFR was expressed by using westernblotting after overexpressing the Δ160 variant as the RON activationform in the Colo320HSR colon cancer cell line in which the RON was notexpressed in order to verify whether the activation of the EGFR proteinwas induced according to actual RON activation.

As a result, as illustrated in FIG. 1F, when the RON was activated, itwas verified that the phosphorylated protein of the EGFR was increased(a), and it was verified that the phosphorylation of the EGFR wasinduced through an RTK-protein array after overexpressing the Δ160variant as the RON activation form. Accordingly, it was seen that theactivity of the EGFR was induced according to the activation of the RON.

Example 2. Change of EGFR Activity according to RON Activity

2-1. Activation of EGFR Protein by Activation of RON Protein

The inventors performed western blot, after overexpressing the Δ160variant as the activation form of the RON gene in the LoVo andColo320HSR colon cancer cell lines in which the RON was not expressed inorder to verify the activation of the EGFR protein by the RONactivation.

As a result, as illustrated in FIG. 2A, it was verified that theactivity of the EGFR was increased according to the RON activation.

2-2. Change in Activity of EGFR Protein by RON Inhibition

The inventors verified the phosphorylation induction of the EGFR byperforming the western blot, after artificially suppressing the RON byusing a siRNA technique in the KM12C and HT29 colon cancer cell lines inwhich the RON was expressed and activated, in order to verify the changein activity of the EGFR protein by the activation of the RON protein.

As a result, as illustrated in FIG. 2B, it was verified that theactivity of the EGFR was also decreased according to the RON inhibition.

2-3. Change in Activity of EGFR Ligand-Dependent EGFR by RON Inhibition

The inventors verified the phosphorylation degree of the EGFR accordingto an EGF ligand treating time which induces the activity of the EGFR,after inhibiting the RON by using the siRNA technique in the KM12C coloncancer cell line in which the RON was expressed and activated in orderto verify the change in the activity of EGFR ligand-dependent EGFR.

As a result, as illustrated in FIG. 2C, it was verified that thephosphorylation of the EGFR induced by the EGF was decreased and theactivity was decreased according to the RON inhibition.

Example 3. Drug Susceptibility Analysis of Cetuximab according toActivation of RON Protein

3-1. Analysis of Binding in Endogenous Cells between RON Protein andEGFR Protein

The inventors observed presence or absence of the binding between theRON protein and the EGFR protein by immunoprecipitation by using the RONantibody in order to analyze the presence or absence of the bindingbetween the RON protein and the EGFR protein in the cells of the CaCO2colon cancer cell line in which the RON was not activated and the KM12Ccolon cancer cell line in which the RON was activated.

As a result, as illustrated in FIG. 3A, it can be seen that in the CaCO2cell in which the RON was not activated, the binding between the RONprotein and the EGFR protein was not made, and in the KM12C cell inwhich the RON was activated, the binding between the RON protein and theEGFR protein was made.

3-2. Analysis of binding in exogenous cells between RON protein and EGFRprotein

The inventors observed presence or absence of the binding between theRON protein and the EGFR protein by immunoprecipitation by using the RONantibody, after respectively overexpressing a RON normal gene and anEGFR normal gene and simultaneously overexpressing the RON normal geneand the EGFR normal gene in the colon cancer cell line CaCO2 in whichthe RON was not activated.

As a result, as illustrated in FIG. 3B, it can be seen that the bindingbetween the RON protein and the EGFR protein was made in the case ofsimultaneously overexpressing the RON normal gene and the EGFR normalgene.

3-3. Analysis of EGFR Protein Domain Bound to RON Protein through inVivo Full Down Assay

The inventors observed a domain of the EGFR bound to the RON proteinthrough immunoprecipitation by using the RON antibody after respectivelyoverexpressing a RON protein, a kinase domain protein of the EGFR, andan extracellular domain (EC) protein and simultaneously overexpressingthe RON and the kinase domain protein of the EGFR in a 293T cell line inwhich the RON and the EGFR were not expressed, not the colon cancer cellline.

As a result, as illustrated in FIG. 3C, it can be seen that the RON andthe EC protein of the EGFR were bound to each other.

3-4. Analysis of Domain of EGFR Protein Bound to RON Protein through inVitro Cell Free Full Down Assay

The inventors observed presence or absence of binding between the RONnormal protein and the active form Δ160 protein by reacting with thekinase domain protein and the EC protein of the EGFR by using an invitro cell free full down assay, in order to re-verify binding betweenthe RON protein and the EC protein of the EGFR.

As a result, as illustrated in FIG. 3D, it can be seen that both the RONnormal protein and the active form Δ160 protein bound to the EC proteinof the EGFR.

3-5. Analysis of Prediction of RON Protein Binding Site Bound to EGFRProtein using computer modeling

The inventors predicted that a binding site of the RON bound to the EGFRby introducing a construct of the EGFR protein and a construct of theRON protein by using a computer modeling system, in order to analyze amain binding site of the RON bound to the EGFR protein.

As a result, as illustrated in FIG. 3E, an E387 site and a H424 site ofthe amino acid of the RON protein were predicted and analyzed as siteswhich may be bound to the EGFR protein.

3-6. Analysis of Binding Site of RON Protein Bound to EGFR Protein

The inventors observed binding between normal form, active form, andmutant form RON proteins and the EGFR protein throughimmunoprecipitation by using the RON antibody after respectivelyoverexpressing the RON normal form gene, the active form Δ160, and E387Aand H424L point mutant genes of the RON and simultaneouslyoverexpressing the EGFR gene in the 293T cell line in which the RON andthe EGFR were not expressed, in order to analyze the main binding siteof the RON bound to the EGFR protein.

As a result, as illustrated in FIG. 3F, the binding was verified in thenormal form, the active form, and the E387A mutant form of the RON, andin the H424L mutant form of the RON, the binding of the EGFR protein wasnot verified.

3-7. Analysis of EGFR Activity according to Binding Site of RON ProteinBound to EGFR Protein

The inventors verified the phosphorylation of the EGFR as the activityof the EGFR through western blot by respectively overexpressing the RONnormal form gene and the active form Δ160 variant and the H424L mutantgene verified in Example 3-6 (see FIG. 3F) and simultaneouslyoverexpressing the EGFR normal form gene in the 293T cell line in whichthe RON and the EGFR were not expressed, in order to analyze the EGFRactivity according to the binding site of the RON bound to the EGFRprotein.

As a result, as illustrated in FIG. 3G, like the EGFR active form mutant(EGFR del19), only in the place simultaneously overexpressing the EGFRnormal form, the active form Δ160 variant of the RON, phosphorylation ofthe EGFR was induced, in the normal form of the RON and the H424L mutantform, the phosphorylation of the EGFR was not induced, and the activityof the EGFR was exhibited according to the activation of the RON.

Example 4. Analysis of Activity of EGFR according to RON Activity

4-1. Analysis of Ligand-Dependent EGFR Activity according to RON ActiveForm Protein

The inventors analyzed the EGFR activity by EGF which is a ligand ofEGFR according to presence of the active form Δ160 protein of the RONafter overexpressing the normal form protein of the EGFR and the kinasedead protein of the EGFR in the 293T cell line in which the RON and theEGFR were not expressed.

As a result, as illustrated in FIG. 4A, in the case where the activeform Δ160 of the RON was not present, only in the normal form protein ofthe EGFR, the activation of the EGFR was verified by EGF, in the casewhere the active form Δ160 protein of the RON was present, theactivation of the EGFR was observed regardless of the EGF and the EGFRkinase dead protein, and thus, it can be seen that the active form Δ160protein of the RON induced the activation of the EGFRligand-independently of the EGFR.

4-2. Analysis of Ligand-Dependent EGFR Activity according toLigand-Dependent Activity of RON

The inventors analyzed the EGFR activity by EGF which is a ligand ofEGFR according to presence of MSP which is a ligand inducing theactivity of the RON after overexpressing the normal form protein of theEGFR and the kinase dead protein of the EGFR in the 293T cell line inwhich the RON and the EGFR were not expressed.

As a result, as illustrated in FIG. 4B, in the case where the MSP as theligand inducing the activity of the RON was not present, only in thenormal form protein of the EGFR, the activation of the EGFR was verifiedby EGF, in the case where the MSP as the ligand inducing the activity ofthe RON was present, the activation of the EGFR was observed regardlessof the EGF and the EGFR kinase dead protein, and thus, it can be seenthat if the RON was the active form by the MSP as the ligand inducingthe activity of the RON, the activation of the EGFR was inducedligand-independently of the EGFR.

Further, as illustrated in FIG. 4C, when RONΔ155/K1114N (kinase activityinhibition form as a kinase dead RON mutant) and EGFR wereoverexpressed, it can be seen that the activity of EGFR was reduced ascompared with a case where only the active form of the RON wasoverexpressed.

Example 5. Analysis of EGFR Activity and Association for Cetuximabaccording to RON Activity

5-1. Analysis of Activity Inhibition of EGFR through RON ActivityInhibition and EGFR Activity Inhibition for Cetuximab

As listed in FIG. 9, it was known that the KM12C colon cancer cell linehad the KRAS, NRAS, and BRAF wild type genes and had resistance tocetuximab (Todd M. et al, Dual Pharmacological Targeting of the MAPKinase and PI3K/mTOR Pathway in Preclinical Models of Colorectal Cancer.PLOS 2014, Volume 9, Issue 11, e113037).

Hereinabove, the inventors verified in Example 1-1 that the RON wasactivated in the KM12C colon cancer cell line.

Accordingly, the activation degree of the EGFR was observed by westernblot by treating cetuximab for each time after artificially inhibitingthe RON by an siRNA technique in the KM12C colon cancer cell line whichhad KRAS, NRAS, and BRAF wild type genes, had resistance to cetuximaband in which the RON was activated, as described above, in order toanalyze the activity inhibition of the EGFR through the RON activityinhibition and the EGFR activity inhibition for cetuximab.

As a result, as illustrated in FIG. 5A, in the KM12C cell line withoutinhibiting the RON, even though cetuximab was treated, the activity ofEGFR was reduced and then increased over the time, while in the KM12Ccell line in which RON was inhibited with siRNA and cetuximab wastreated, it was verified that the activity of the EGFR was rapidlyreduced and then not increase, and therefore, it can be seen that theactivity of the EGFR for cetuximab was regulated according to thepresence of the RON.

5-2. Analysis of Cell Death of Cetuximab according to Presence of RONActivity

As listed in Table 6, it was known that the CaCo-2 colon cancer cellline had the

TABLE 6 Cell line KRAS BRAF CACO-2* wild-type wild-type SW48* wild-typewild-type HT-29* wild-type mutation at exon 15 (V600E) Colo205*wild-type mutation at exon 15 (V600E) SW480** mutation at exon 2 (G12V)wild-type SW620* mutation at exon 2 (G12V) wild-type RKO* wild-typemutation at exon 15 (V600E) LS174T* mutation at exon 2 (G12V) wild-typeHCT-116* mutation at exon 2 (G12D) wild-typeKRAS, NRAS, and BRAF wild type genes and had susceptibility to cetuximab(Giovanni B. et al, Antitumoral Efficacy of the Protease InhibitorGabexate Mesilate in Colon Cancer Cells Harbouring KRAS, BRAF and PIK3CAMutations. PLOS 2012, Volume 7, Issue 7, e41347).

Hereinabove, the inventors verified in Example 1-1 that the RON was notactivated in the CaCo-2 colon cancer cell line.

Accordingly, in order to analyze the cell death degree according to theRON activity when cetuximab was treated, as described above, in order toverify the cetuximab reactivity in the CaCo-2 colon cancer cell line andthe LIM1215 cell line which had KRAS, NRAS, and BRAF wild type genes andhad susceptibility to cetuximab and in which the RON was not activated,and the KM12C colon cancer cell line in which the RON was activated andwhich had the resistance to cetuximab, the cell death was analyzed bytrypan blue cell counting.

As a result, as illustrated in FIG. 5B, in the CaCo-2 cell line and theLIM1215 cell line in which the RON was not activated, the cell death bycetuximab was observed, while in the KM12C cell line in which the RONwas activated, it can be seen that the cell death by cetuximab was notobserved and the KM12C cell line had the resistance to cetuximab.

5-3. Analysis of Cell Death by Cetuximab after Inhibiting RON Expression

The inventors analyzed the cell death by trypan blue cell counting bytreating cetuximab after artificially inhibiting the RON by the siRNAtechnique in the KM12C colon cancer cell line having the resistance tocetuximab in which the RON was activated.

As a result, as illustrated in FIG. 5C, in the case of inhibiting theRON, it can be seen that the susceptibility to cetuximab was increasedand the cell death was increased.

5-4. Analysis of Sub-Signaling Mechanism by Cetuximab after InhibitingRON Expression

The inventors observed a sub-signaling mechanism through western blot bytreating cetuximab after artificially inhibiting the RON by the siRNAtechnique in the KM12C colon cancer cell line having the resistance tocetuximab in which the RON was activated.

As a result, as illustrated in FIG. 5D, in the group in which theexpression of the RON was inhibited and cetuximab was treated, it can beseen that a p-ERK protein that played an important role in cell survivalwas reduced and cleaved caspase-3 as an apoptosis marker protein wasincreased.

5-5. Analysis of Cell Death By Cetuximab after Inhibiting RON Activity

The inventors analyzed the cell death by trypan blue cell counting inthe group in which an inhibitor LY2801653 capable of inhibiting theactivity of the RON and cetuximab were treated, respectively, and thegroup in which the LY2801653 and cetuximab were treated together, in theKM12C colon cancer cell line having resistance to cetuximab in which theRON was activated.

As a result, as illustrated in FIG. 5E, in the group in which theLY2801653, RON activity inhibitor and cetuximab were treated together,it can be seen that the cell death was increased.

5-6. Analysis of Cell Death by Cetuximab according to Activity of RON

The inventors observed the cell death for cetuximab after overexpressingΔ160 and Δ155 as the active forms of the RON in the CaCo-2 and SW48colon cancer cell lines having the susceptibility to cetuximab in whichthe RON was not activated.

As a result, as illustrated in FIG. 5F, in the case of overexpressingthe Δ160 and Δ155 as the active forms of the RON in the CaCo-2 and SW48cell lines which had the susceptibility to cetuximab, it can be seenthat the cell death was inhibited by cetuximab and the resistance tocetuximab was exhibited.

5-7. Analysis of Cell Growth Inhibition by Cetuximab according toActivity of RON

The inventors observed the cell growth for cetuximab by a colonyformation method by overexpressing the Δ160 variant as the active formof the RON in the CaCo-2 colon cancer cell line having thesusceptibility to cetuximab in which the RON was not activated.

As a result, as illustrated in FIG. 5G, in a parent CaCo-2 cell line ofwhich the number of colonies was reduced by cetuximab, when the Δ160variant as the active form of the RON was overexpressed, it can be seenthat even though cetuximab was treated, the number of colonies was notalmost reduced and the resistance of the cell growth to cetuximab wasexhibited by the Δ160 protein as the active form of the RON.

5-8. Analysis of Cell Death by Cetuximab in RON Knockout Isogenic CellLine

The inventors observed the cell death by treating cetuximab for eachconcentration in #1 clone with good RON knockout efficiency, in order toanalyze the cell death and the cell growth for cetuximab by preparing acell line in which the RON gene was knockout by using a CRISPR/Cas9method in the KM12C colon cancer cell line having resistance tocetuximab in which the RON was activated.

As a result, as illustrated in FIG. 5H, it was verified that the celldeath was increased as compared with the parent cell line, and whenknockoutting the RON, it can be seen that the susceptibility tocetuximab was increased.

5-9. Analysis of Cell Growth Inhibition by Cetuximab in RON KnockoutIsogenic Cell Line

The inventors observed the cell growth for cetuximab by a colonyformation method by using a cell line in which the RON gene wasknocked-out by using the CRISPR/Cas9 method in the KM12C colon cancercell line having resistance to cetuximab in which the RON was activated.

As a result, as illustrated in FIG. 5I, it was verified that the numberof colonies decreased as compared with the parent cell line, and whenknockoutting the RON, it can be seen that the susceptibility tocetuximab was increased.

Example 6. Analysis of Tumor Inhibition Effect by Cetuximab according toRON in In Vivo Xenograft Model using RON Knockout Isogenic Cell Line

The inventors observed the tumor inhibition effect by cetuximab in an invivo xenograft model by using a cell line in which the RON gene wasknocked-out by using the CRISPR/Cas9 method in the KM12C colon cancercell line having resistance to cetuximab in which the RON was activated.

As a result, as illustrated in FIG. 6, as compared with the parent cellline, in the RON knockout xenograft mouse, it was observed that the sizeof the tumor was significantly reduced, and when the activation of theRON was knocked-out, it can be seen that the in vivo susceptibility bycetuximab was increased.

Accordingly, the activation of the RON exhibits possibility as thebiomarker for predicting the susceptibility to cetuximab as theEGFR-targeted agent.

*Example 7. Verification of Cell Death Induction by Treating RONAntibody Anticancer Agent and Cetuximab Together

The inventors verified the cell death induction by treating an RONantibody anticancer agent and cetuximab together.

As a result, as illustrated in FIG. 7, when treating narnatumab andcetuximab which were antibody anticancer agents targeting the RONtogether, it was verified that the cell death was induced. In this case,when the change in expression of proteins was verified, it was verifiedthat the activity of the EGFR and the RON was inhibited and the activityof the ERK was inhibited.

Example 8. Analysis Relationship between Cetuximab Reactivity and RONActivity

The inventors analyzed a relationship between cetuximab reactivity andRON activity.

As a result, as illustrated in FIG. 8, the colon cancer patients wereverified as having RON active forms (p-RON positive) and EGFR activeforms (P-EGFR positive) (see left graph). Further, it was verified thatthe patients prescribed with cetuximab among the colon cancer patients(double positive) had different susceptibility to cetuximab according tothe RON activity.

1. A composition for predicting susceptibility to an epidermal growthfactor receptor (EGFR)-targeted agent, the composition comprising anagent which measures an expression level of RON (recepteur d' originenantais, Gene Bank accession No. NM_002447.1) gene; or an expression oractivity level of a protein thereof.
 2. The composition of claim 1,wherein the composition further comprises an agent which measures anexpression level of one or more genes selected from the group consistingof KRAS (V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog, Gene Bankaccession No. NM_033360.2), NRAS (neuroblastoma RAS viral (v-ras)oncogene homolog, Gene Bank accession No. NP_002515.1), BRAF (v-rafmurine sarcoma viral oncogene homolog B, Gene Bank accession No.NP_004324.2), EGFR (Epidermal Growth Factor Receptor, Gene Bankaccession No. U48722.1), Adam11 (ADAM metallopeptidase domain 11, GeneBank accession No. NM_002390.4), Adam32 (ADAM metallopeptidase domain32, Gene Bank accession No. NM_145004.5), FZD4 (Frizzled family receptor4, Gene Bank accession No. NM_012193.2), GPER (G protein-coupledestrogen receptor 1, NM_001505.2), and GPR101 (G protein-coupledreceptor 101, Gene Bank accession No. NM_054021.1) genes; or anexpression or activity level of a protein of the genes.
 3. Thecomposition of claim 2, wherein the KRAS (V-Ki-ras2 Kirsten rat sarcomaviral oncogene homolog, Gene Bank accession No. NM_033360.2), NRAS(neuroblastoma RAS viral (v-ras) oncogene homolog, Gene Bank accessionNo. NP_002515.1), or BRAF (v-raf murine sarcoma viral oncogene homologB, Gene Bank accession No. NP_004324.2) gene is a wild type.
 4. Thecomposition of claim 1, wherein the EGFR-targeted agent is a therapeuticagent for one or more selected from the group consisting ofadrenocorticotropic hormone (ACTH) produced tumors, acute lymphocytic orlymphoblastic leukemia, acute or chronic lymphocytic leukemia, acutenon-lymphocytic leukemia, bladder cancer, brain tumors, breast cancer,cervix cancer, chronic myelogenous leukemia, lymphomas, endometriosis,esophagus cancer, bladder cancer, Ewing's sarcoma, tongue cancer,Hopkins lymphoma, Capo sheath sarcoma, kidney cancer, liver cancer, lungcancer, mesothelioma, multiple myeloma, neuroblastoma, non-hopkinlymphoma, osteosarcoma, ovarian cancer, lobular carcinoma, prostatecancer, pancreatic cancer, colorectal cancer, penis cancer,retinoblastoma, skin cancer, stomach cancer, thyroid cancer, uterinecancer, testicular cancer, wilms tumor, and trophoblastoma.
 5. Thecomposition of claim 1, wherein the EGFR-targeted agent is one or moreselected from the group consisting of cetuximab, gefitinib, erlotinib,panitumumab, PKI-166, EKB-569, HKI-272 (WAY-177820), icotinib,brigatinib, afatinib, lapatinib, canertinib, AEE788, XL647, and Zactima.6. The composition of claim 1, wherein the agent which measures theexpression level of the gene comprises antisense oligonucleotide, aprimer pair, or a probe which is specifically bound to mRNA of the gene.7. The composition of claim 1, wherein the agent which measures theexpression or activity level of the protein comprises an antibody, apeptide, or a nucleotide which is specifically bound to the protein. 8.The composition of claim 1, wherein when the expression level of the RONgene; or the expression or activity level of the protein thereof is lowas compared with a normal level, it is determined that there is thesusceptibility to the EGFR-targeted agent.
 9. The composition of claim1, wherein when the expression level of the RON gene; or the expressionor activity level of the protein thereof is high as compared with anormal level, it is determined that there is the resistance to theEGFR-targeted agent.
 10. A kit for predicting susceptibility to an EGFR(epidermal growth factor receptor)-targeted agent, the kit comprisingthe composition of claim 1.